Methods for detecting vitamin D metabolites

ABSTRACT

Provided are methods of detecting the presence or amount of a vitamin D metabolite in a sample using mass spectrometry. The methods generally comprise ionizing a vitamin D metabolite in a sample and detecting the amount of the ion to determine the presence or amount of the vitamin D metabolite in the sample. Also provided are methods to detect the presence or amount of two or more vitamin D metabolites in a single assay.

FIELD OF THE INVENTION

The invention relates to the detection of vitamin D metabolites. In aparticular aspect, the invention relates to methods for detectingvitamin D metabolites by mass spectrometry.

BACKGROUND OF THE INVENTION

Vitamin D is an essential nutrient with important physiological roles inthe positive regulation of calcium (Ca²⁺) homeostasis. Vitamin D can bemade de novo in the skin by exposure to sunlight or it can be absorbedfrom the diet. There are two forms of vitamin D; vitamin D₂(ergocalciferol) and vitamin D₃ (cholecalciferol). Vitamin D₃ is theform synthesized de novo by animals. It is also a common supplementedadded to milk products and certain food products produced in the UnitedStates. Both dietary and intrinsically synthesized vitamin D₃ mustundergo metabolic activation to generate the bioactive metabolites. Inhumans, the initial step of vitamin D₃ activation occurs primarily inthe liver and involves hydroxylation to form the intermediate metabolite25-hydroxycholecalciferol (calcifediol; 25OHD₃). Calcifediol is themajor form of Vitamin D₃ in the circulation. Circulating 25OHD₃ is thenconverted by the kidney to form 1,25-dihydroxyvitamin D₃ (calcitriol;1,25(OH)₂D₃), which is generally believed to be the metabolite ofVitamin D₃ with the highest biological activity.

Vitamin D₂ is derived from fungal and plant sources. Manyover-the-counter dietary supplements contain ergocalciferol (vitamin D₂)rather than cholecalciferol (vitamin D₃). Drisdol, the only high-potencyprescription form of vitamin D available in the United States, isformulated with ergocalciferol. Vitamin D₂ undergoes a similar pathwayof metabolic activation in humans as Vitamin D₃, forming the metabolites25OHD₂ and 1,25(OH)₂D₂. Vitamin D₂ and vitamin D₃ have long been assumedto be biologically equivalent in humans, however recent reports suggestthat there may be differences in the bioactivity and bioavailability ofthese two forms of vitamin D (Armas et. al., (2004) J. Clin. Endocrinol.Metab. 89:5387-5391).

Measurement of vitamin D, the inactive vitamin D precursor, is rare inclinical settings and has little diagnostic value. Rather, serum levelsof 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂ (total25-hydroxyvitamin D; “25OHD”) are a useful index of vitamin Dnutritional status and the efficacy of certain vitamin D analogs.Therefore, the measurement of 25OHD is commonly used in the diagnosisand management of disorders of calcium metabolism. In this respect, lowlevels of 25OHD are indicative of vitamin D deficiency associated withdiseases such as hypocalcemia, hypophosphatemia, secondaryhyperparathyroidism, elevated alkaline phosphatase, osteomalacia inadults and rickets in children. In patients suspected of vitamin Dintoxication, elevated levels of 25OHD distinguishes this disorder fromother disorders that cause hypercalcemia.

Measurement of 1,25(OH)₂D is also used in clinical settings, however,this test has a more limited diagnostic usefulness than measurements of25OHD. Factors that contribute to limitations of the diagnostic valuesof 1,25(OH)₂D as an index of Vitamin D status include the precision ofthe endogenous regulation of renal production of the metabolite and itsshort half-life in circulation. However, certain disease states can bereflected by circulating levels of 1,25(OH)₂D, for example kidneydisease and kidney failure often result in low levels of 1,25(OH)₂D.Elevated levels of 1,25(OH)₂D may be indicative of excess parathyroidhormone or can be indicative of certain diseases such as sarcoidosis orcertain types of lymphomas.

Detection of vitamin D metabolites has been accomplished byradioimmunoassay with antibodies co-specific for 25OHD₂ and 25OHD₃.Because the current immunologically-based assays do not separatelyresolve 25OHD₂ and 25OHD₃, the source of any deficiency nutritional ofvitamin D cannot be determined without resorting to other tests. Morerecently, reports have been published that disclose methods fordetecting specific Vitamin D metabolites using mass spectrometry. Forexample Yeung B, et al., J Chromatogr. 1993, 645(1):115-23; Higashi T,et al., Steroids. 2000, 65(5):281-94; Higashi T, et al., Biol PharmBull. 2001, 24(7):738-43; and Higashi T, et al., J Pharm Biomed Anal.2002, 29(5):947-55 disclose methods for detecting various vitamin Dmetabolites using liquid chromatography and mass spectrometry. Thesemethods require that the metabolites be derivatized prior to detectionby mass-spectometry. Methods to detect underivatized 1,25(OH)₂D₃ byliquid chromatography/mass-spectrometry are disclosed in Kissmeyer andSonne, J Chromatogr A. 2001, 935(1-2):93-103.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the presence oramount of a vitamin D metabolite in a test sample by mass spectrometry,including tandem mass spectometry. Preferably, the methods of theinvention do not include derivatizing the sample or the vitamin Dmetabolites prior to the mass spectrometry analysis.

In one aspect the invention provides a method for determining thepresence or amount of 25-hydroxyvitamin D in a biological sample. Themethod may include: (a) ionizing 25-hydroxyvitamin D, if present in thesample; and (b) detecting the presence or amount of the ion by massspectrometry, wherein the presence or amount of the ion is related tothe presence or amount of 25-hydroxyvitamin D in the test sample. Insome preferred embodiments, the ionization step (a) may include (i)ionizing 25-hydroxyvitamin D, if present in the sample, to produce a25-hydroxyvitamin D ion; (ii) isolating the 25-hydroxyvitamin D ion bymass spectrometry to provide a precursor ion; and (iii) effecting acollision between the isolated precursor ion and an inert collision gasto produce at least one fragment ion detectable in a mass spectrometer.In certain embodiments the 25-hydroxyvitamin D is 25-hydroxyvitamin D₃,in other related embodiments the 25-hydroxyvitamin D is25-hydroxyvitamin D₂.

In another aspect, the invention provides a method for determining thepresence or amount of 1,25-dihydroxyvitamin D₂ in a biological sample.The method may include: (a) ionizing the purified 1,25-dihydroxyvitaminD₂, if present in the sample, and (b) detecting the presence or amountof the ion by mass spectrometry, wherein the presence or amount of theion is related to the presence or amount of 1,25-dihydroxyvitamin D₂ inthe test sample. In certain embodiments, the ionization step (a) mayinclude (i) ionizing 1,25-dihydroxyvitamin D₂, if present in the sampleto produce a 1,25-dihydroxyvitamin D₂ ion; (ii) isolating the1,25-dihydroxyvitamin D₂ ion by mass spectometry to provide a precursorion; and (iii) effecting a collision between the isolated precursor ionand an inert collision gas to produce at least one fragment iondetectable in a mass spectrometer.

In another aspect the invention provides a method for determining thepresence or amount of two or more vitamin D metabolites in a test samplein a single assay. The method includes ionizing the vitamin Dmetabolites to generate ions specific for each of the vitamin Dmetabolites of interest and detecting the presence or amount of the ionsby mass spectrometry, wherein the presence or amount of the ions isrelated to the presence or amount of the vitamin D metabolites in thetest sample. Preferably, the method does not involve derivatizing thesamples or the vitamin D metabolites prior to analysis by massspectrometry. In certain embodiments the mass spectrometry analysis ofthe method is tandem mass spectrometry.

As used herein, the term “vitamin D metabolite” refers to any vitamin Danalog or any chemical species related to vitamin D. Vitamin Dmetabolites may include analogs of, or a chemical species related to,vitamin D₂ or vitamin D₃. Vitamin D metabolites may be found in thecirculation of an animal and/or may be generated by a biologicalorganism, such as an animal, or by biotransformation of vitamin D₂ orvitamin D₃. Vitamin D metabolites may be metabolites of naturallyoccurring forms of vitamin D or may be metabolites of synthetic vitaminD analogs. In certain embodiments a vitamin D metabolite is one or morecompounds selected from the group consisting of 25-hydroxyvitamin D₃,25-hydroxyvitamin D₂, 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitaminD₂.

Purification in the context of the methods of the invention does notrefer to removing all materials from the sample other than theanalyte(s) of interest. Instead, purification refers to a procedure thatenriches the amount of one or more analytes of interest relative to oneor more other components of the sample. In preferred embodiments,purification can be used to remove one or more interfering substances,e.g., one or more substances that would interfere with detection of ananalyte ion by mass spectrometry.

As used herein, “biological sample” refers to any sample from abiological source. As used herein, “body fluid” means any fluid that canbe isolated from the body of an individual. For example, “body fluid”may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, “derivatizing” means reacting two molecules to form anew molecule. Derivatizing agents may include isothiocyanate groups,dinitro-fluorophenyl groups, nitrophenoxycarbonyl groups, and/orphthalaldehyde groups.

As used herein, “chromatography” refers to a process in which a chemicalmixture carried by a liquid or gas is separated into components as aresult of differential distribution of the solutes as they flow aroundor over a stationary liquid or solid phase.

As used herein, “liquid chromatography” (LC) means a process ofselective retardation of one or more components of a fluid solution asthe fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). “Liquid chromatography”includes reverse phase liquid chromatography (RPLC), high performanceliquid chromatography (HPLC) and high turbulence liquid chromatography(HTLC).

As used herein, the term “HPLC” or “high performance liquidchromatography” refers to liquid chromatography in which the degree ofseparation is increased by forcing the mobile phase under pressurethrough a stationary phase, typically a densely packed column.

As used herein, the term “gas chromatography” refers to chromatographyin which the sample mixture is vaporized and injected into a stream ofcarrier gas (as nitrogen or helium) moving through a column containing astationary phase composed of a liquid or a particulate solid and isseparated into its component compounds according to the affinity of thecompounds for the stationary phase

As used herein, “mass spectrometry” (MS) refers to an analyticaltechnique to identify compounds by their mass. MS technology generallyincludes (1) ionizing the compounds and potentially fractionating thecompounds to form charged compounds; and (2) detecting the molecularweight of the charged compound and calculating a mass-to-charge ratio(m/z). The compound may be ionized and detected by any suitable means. A“mass spectrometer” generally includes an ionizer and an ion detector.

The term “electron ionization” as used herein refers to methods in whichan analyte of interest in a gaseous or vapor phase interacts with a flowof electrons. Impact of the electrons with the analyte produces analyteions, which may then be subjected to a mass spectrometry technique.

The term “chemical ionization” as used herein refers to methods in whicha reagent gas (e.g. ammonia) is subjected to electron impact, andanalyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

The term “fast atom bombardment” as used herein refers to methods inwhich a beam of high energy atoms (often Xe or Ar) impacts anon-volatile test sample, desorbing and ionizing molecules contained inthe sample. Test samples are dissolved in a viscous liquid matrix suchas glycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crownether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine.

The term “field desorption” as used herein refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

The term “ionization” as used herein refers to the process of generatingan analyte ion having a net electrical charge equal to one or moreelectron units. Negative ions are those having a net negative charge ofone or more electron units, while positive ions are those having a netpositive charge of one or more electron units.

The term “operating in negative ion mode” refers to those massspectrometry methods where negative ions are detected. Similarly,“operating in positive ion mode” refers to those mass spectrometrymethods where positive ions are detected.

The term “desorption” as used herein refers to the removal of an analytefrom a surface and/or the entry of an analyte into a gaseous phase.

The term “about” as used herein in reference to quantitativemeasurements, refers to the indicated value plus or minus 10%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the linearity of the quantification of 25OHD₂ in samplesserially diluted with the 25OHD diluent using the LC-MS/MS assay.

FIG. 2 shows the linearity of the quantification of 25OHD₃ in samplesserially diluted with the 25OHD diluent using the LC-MS/MS assay.

FIG. 3 shows the linearity of the quantification by LC-MS/MS of seriallydiluted samples spiked with of 25OHD₂ and 25OHD₃ to final concentrationsof 512 ng/mL.

FIG. 4 shows the correlation between detection of total25-hydroxyvitamin D using the LC-MS/MS method and a commerciallyavailable radioimmunoassay kit.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are methods for detecting the presence or amount of one ormore vitamin D metabolites in a test sample. In certain aspects themethod involves ionizing the vitamin D metabolites and detecting the ionby mass spectrometry, wherein the presence of the ion is related to thepresence or amount of the vitamin D metabolite in the test sample. Inrelated aspects, the method may include (a) purifying a vitamin Dmetabolite, if present in the test sample, by chromatography, (b)ionizing the purified vitamin D metabolite and (c) detecting thepresence or amount of the ion, wherein the presence or amount of the ionis related to the presence or amount of the vitamin D metabolite in thetest sample. In preferred embodiments, the ionizing step (b) maycomprise (i) ionizing a vitamin D metabolite, if present in the sample,to produce an ion; (ii) isolating the vitamin D metabolite ion by massspectometry to provide a precursor ion; and (iii) effecting a collisionbetween the isolated precursor ion and an inert collision gas to produceat least one fragment ion detectable in a mass spectrometer. In certainembodiments at least one fragment ion is detected, wherein the presenceor amount of the fragment ion is related to the presence or amount ofthe vitamin D metabolite in the test sample. In some embodiments, themethods of the invention can be used to detect and quantify two or morevitamin D metabolites in a single assay.

Suitable test samples include any sample that might contain the analyteof interest and/or one or more metabolites or precursors thereof. Forexample, samples obtained during the manufacture of an analyte can beanalyzed to determine the composition and yield of the manufacturingprocess. In certain embodiments, a sample is a biological sample; thatis, a sample obtained from any biological source, such as an animal, acell culture, an organ culture, etc. Particularly preferred are samplesobtained from a human, such as a blood, plasma, serum, hair, muscle,urine, saliva, tear, cerebrospinal fluid, or other tissue sample. Suchsamples may be obtained, for example, from a patient; that is, a livingperson presenting themselves in a clinical setting for diagnosis,prognosis, or treatment of a disease or condition.

Samples may be processed or purified to obtain preparations that aresuitable for the desired type of chromatography and/or for analysis bymass spectrometry. Various procedures may be used for this purposedepending on the type sample or the type of chromatography. Examplesinclude filtration, extraction, precipitation, centrifugation, dilution,combinations thereof and the like. Protein precipitation is onepreferred method of preparing a liquid biological sample, such as serumor plasma, for chromatography. In a preferred embodiment, one volume ofthe liquid sample is added to four volumes of methanol. This results inthe precipitation of most protein while vitamin D metabolites are fullysoluble in the resulting supernatant. The samples can then becentrifuged to separate the liquid supernatant from the pellet. Theresultant supernatants can then be applied to liquid chromatography andmass spectrometry analysis. Preferably, sample preparation does notinvolve the use of a derivitization agent. Therefore the methods of theinvention preferably do not include a derivatization step prior toanalysis of the sample by mass spectrometry. Thus, the methods allow thedetection and quantification directly of ions of the desired vitamin Dmetabolites rather than ions of a derivatized form of the vitamin Dmetabolites.

The sample, or the processed sample, may be purified prior to analysisby mass spectrometry. Such purification, or sample clean-up, refers to aprocedure that enriches of one or more analytes of interest relative toone or more other components of the sample. Typically, chromatography,preferably liquid chromatography, more preferably high performanceliquid chromatography is used for the purification. In preferredembodiments the chromatography is not gas chromatography. Preferably,the methods of the invention are performed without subjecting the testsamples, or the vitamin D metabolites of interest, to gas chromatographyprior to mass spectrometric analysis.

Various methods have been described involving the use of HPLC for sampleclean-up prior to mass spectrometry analysis. See, e.g., Taylor et al.,Therapeutic Drug Monitoring 22:608-12 (2000) (manual precipitation ofblood samples, followed by manual C18 solid phase extraction, injectioninto an HPLC for chromatography on a C18 analytical column, and MS/MSanalysis); and Salm et al., Clin. Therapeutics 22 Supl. B:B71-B85 (2000)(manual precipitation of blood samples, followed by manual C18 solidphase extraction, injection into an HPLC for chromatography on a C18analytical column, and MS/MS analysis). One of skill in the art canselect HPLC instruments and columns that are suitable for use in theinvention. The chromatographic column typically includes a medium (i.e.,a packing material) to facilitate separation of chemical moieties (i.e.,fractionation). The medium may include minute particles. The particlesinclude a bonded surface that interacts with the various chemicalmoieties to facilitate separation of the chemical moieties such asvitamin D metabolites. One suitable bonded surface is a hydrophobicbonded surface such as an alkyl bonded surface. Alkyl bonded surfacesmay include C-4, C-8, or C-18 bonded alkyl groups, preferably C-18bonded groups. The chromatographic column includes an inlet port forreceiving a sample and an outlet port for discharging an effluent thatincludes the fractionated sample. In the method, the test sample isapplied to the column at the inlet port, eluted with a solvent orsolvent mixture, and discharged at the outlet port. Different solventmodes may be selected for eluting the analytes. For example, liquidchromatography may be performed using a gradient mode, an isocraticmode, or a polytyptic (i.e. mixed) mode. In preferred embodiments, HPLCis performed on a multiplexed analytical HPLC system with a C18 solidphase using isocratic separation with 100% methanol as the mobile phase.

Recently, high turbulence liquid chromatography (“HTLC”), also calledhigh throughput liquid chromatography, has been applied for samplepreparation prior to analysis by mass spectrometry. See, e.g., Zimmer etal., J. Chromatogr. A 854:23-35 (1999); see also, U.S. Pat. Nos.5,968,367; 5,919,368; 5,795,469; and 5,772,874, each of which is herebyincorporated by reference in its entirety. Traditional HPLC analysisrelies on column packings in which laminar flow of the sample throughthe column is the basis for separation of the analyte of interest fromthe test sample. The skilled artisan will understand that separation insuch columns is a diffusional process. In contrast, it is believed thatturbulent flow, such as that provided by HTLC columns and methods, mayenhance the rate of mass transfer, improving the separationcharacteristics provided. In some embodiments, high turbulence liquidchromatography (HTLC), alone or in combination with one or morepurification methods, may be used to purify the vitamin D metabolite ofinterest. In such embodiments samples may be extracted using an HTLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column prior to ionization. Because thesteps involved in these two HTLC procedures can be linked in anautomated fashion, the requirement for operator involvement during thepurification of the analyte can be minimized.

The terms “mass spectrometry” or “MS” as used herein refer to methods offiltering, detecting, and measuring ions based on their mass-to-chargeratio, or “m/z.” In general, one or more molecules of interest, such avitamin D metabolites, are ionized and the ions are subsequentlyintroduced into a mass spectrographic instrument where, due to acombination of magnetic and electric fields, the ions follow a path inspace that is dependent upon mass (“m”) and charge (“z”). See, e.g.,U.S. Pat. No. 6,204,500, entitled “Mass Spectrometry From Surfaces;”U.S. Pat. No. 6,107,623, entitled “Methods and Apparatus for Tandem MassSpectrometry;” U.S. Pat. No. 6,268,144, entitled “DNA Diagnostics BasedOn Mass Spectrometry;” U.S. Pat. No. 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis21:1164-67 (2000), each of which is hereby incorporated by reference inits entirety, including all tables, figures, and claims.

The mass spectrometer will include an ion source for ionizing thefractionated sample and creating charged molecules for further analysis.For example ionization of the sample may be performed by electrosprayionization (ESI), atmospheric pressure chemical ionization (ACPI),photoinonization, electron ionization, fast atom bombardment(FAB)/liquid secondary ionization (LSIMS), matrix assisted laserdesorption ionization (MALDI), field ionization, field desorption,thermospray/plasmaspray ionization, and particle beam ionization. Theskilled artisan will understand that the choice of ionization method canbe determined based on the analyte to be measured, type of sample, thetype of detector, the choice of positive versus negative mode, etc.

After the sample has been ionized, the positively charged or negativelycharged ions thereby created may be analyzed to determine amass-to-charge ratio (i.e., m/z). Preferably, the negatively chargedions are analyzed. Suitable analyzers for determining mass-to-chargeratios include quadropole analyzers, ion traps analyzers, andtime-of-flight analyzers. The ions may be detected by using severaldetection modes. For example, selected ions may be detected (i.e., usinga selective ion monitoring mode (SIM)), or alternatively, ions may bedetected using a scanning mode. Preferably, the mass-to-charge ratio isdetermined using a quadropole analyzer. For example, in a “quadrupole”or “quadrupole ion trap” instrument, ions in an oscillating radiofrequency field experience a force proportional to the DC potentialapplied between electrodes, the amplitude of the RF signal, and m/z. Thevoltage and amplitude can be selected so that only ions having aparticular m/z travel the length of the quadrupole, while all other ionsare deflected. Thus, quadrupole instruments can act as both a “massfilter” and as a “mass detector” for the ions injected into theinstrument.

One can often enhance the resolution of the MS technique by employing“tandem mass spectrometry,” or “MS/MS.” In this technique, a first, orparent, or precursor, ion generated from a molecule of interest can befiltered in an MS instrument, and these parent ions subsequentlyfragmented to yield one or more second, or daughter, or fragment, ionsthat are then analyzed in a second MS procedure. By careful selection ofparent ions, only ions produced by certain analytes are passed to thefragmentation chamber, where collision with atoms of an inert gas toproduce these daughter ions. Because both the parent and daughter ionsare produced in a reproducible fashion under a given set ofionization/fragmentation conditions, the MS/MS technique can provide anextremely powerful analytical tool. For example, the combination offiltration/fragmentation can be used to eliminate interferingsubstances, and can be particularly useful in complex samples, such asbiological samples.

Additionally, recent advances in technology, such as matrix-assistedlaser desorption ionization coupled with time-of-flight analyzers(“MALDI-TOF”) permit the analysis of analytes at femtomole levels invery short ion pulses. Mass spectrometers that combine time-of-flightanalyzers with tandem MS are also well known to the artisan.Additionally, multiple mass spectrometry steps can be combined inmethods known as “MS/MS^(n).” Various other combinations may beemployed, such as MS/MS/TOF, MALDI/MS/MS/TOF, or SELDI/MS/MS/TOF massspectrometry.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each m/z over a given range (e.g., 100 to1000 amu). The results of an analyte assay, that is, a mass spectrum,can be related to the amount of the analyte in the original sample bynumerous methods known in the art. For example, given that sampling andanalysis parameters are carefully controlled, the relative abundance ofa given ion can be compared to a table that converts that relativeabundance to an absolute amount of the original molecule. Alternatively,molecular standards can be run with the samples, and a standard curveconstructed based on ions generated from those standards. Using such astandard curve, the relative abundance of a given ion can be convertedback into an absolute amount of the original molecule. Numerous othermethods for relating the presence or amount of an ion to the presence oramount of the original molecule will be well known to those of ordinaryskill in the art.

One or more steps of the methods of the invention can be performed usingautomated machines. In certain embodiments, one or more purificationsteps are performed on line, and more preferably all of the purificationand mass spectroscopy steps may be performed in an on line fashion.

In a particularly preferred embodiment vitamin D metabolites using MS/MSas follows. The flow of liquid solvent from a chromatographic column,possibly comprising one or more vitamin D metabolites, enters the heatednebulizer interface of a LC-MS/MS analyzer and the solvent/analytemixture is converted to vapor in the heated tubing of the interface. Theanalytes (i.e. vitamin D metabolites), contained in the nebulizedsolvent, are ionized by the corona discharge needle of the interface,which applies a large voltage to the nebulized solvent/analyte mixture.The ions pass through the orifice of the instrument and enter the firstquadrupole. Quadrupoles 1 and 3 (Q1 and Q3) are mass filters, allowingselection of ions based on their mass to charge ratio (m/z). Quadrupole2 (Q2) is the collision cell, where ions are fragmented. The firstquadrupole of the mass spectrometer (Q1) selects for molecules with themass to charge ratios of the specific vitamin D metabolites to beanalyzed. Ions with the correct m/z ratios of the vitamin D metabolitesare allowed to pass into the collision chamber (Q2), while unwanted ionswith any other m/z collide with the sides of the quadrupole and areeliminated. Ions entering Q2 collide with neutral Argon gas moleculesand fragment. This process is called Collision Activated Dissociation(CAD). The fragment ions generated are passed into quadrupole 3 (Q3),where the fragment ions of the desired vitamin D metabolites areselected while other ions are eliminated. As ions collide with thedetector they produce a pulse of electrons that are converted to adigital signal. In some embodiments the mass/charge ratio (m/z) for the25-hydroxyvitamin D₃ precursor ion is about 383.16, the m/z for the25-hydroxyvitamin D₃ fragment ion is about 211.35, the m/z for the25-hydroxyvitamin D₂ precursor ion is about 395.30 and the m/z for the25-hydroxyvitamin D₂ fragment ions are about 179.1, 209.20, 251.30. Inembodiments where the samples are spiked with hexadeuterated 25OHD₃,⁶D-25OHD₃, for use as an internal standard the mass/charge ratio (m/z)for the ⁶D-25OHD₃ precursor ion is about 389.2 and the m/z for the25-hydroxyvitamin D₃ fragment ion is about 211.30. Mass spectrometryinstruments can vary slightly in determining the mass of a givenanalyte. Thus, the term “about” in the context of mass of an ion or them/z of an ion refers to ±0.5 atomic mass units. The acquired data isrelayed to a computer, which plots counts of the ions collected versustime. The resulting mass chromatograms are similar to chromatogramsgenerated in traditional HPLC methods. The areas under the peaks aredetermined and calibration curves are constructed by plotting standardconcentration versus peak area ratio of analyte/internal standard. Usingthe calibration curves, the concentrations of the vitamin D metabolitesare quantified in the samples.

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

EXAMPLES Example 1 Determination of 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ by LC-MS/MS

Using a Perkin-Elmer MultiProbe II (S/N 432400) robotic liquid handler,human serum samples were first extracted using a protein precipitationmethod by adding 42.5 μl of serum of serum 170 μl of methanol (1:4 ratioof serum:methanol) in a 96-well plate format. For validation-relatedexperiments, the methanol was spiked with hexadeuterated 25OHD₃(⁶D-25OHD₃) as an internal standard. The 96 well plates were centrifugedwhich resulted in the precipitation of most protein in a pellet, whilethe vitamin D metabolites remained in solution in the supernatant. Thesupernatants were then transferred to an HPLC autosampler for loading tothe LC-MS/MS analyzer.

LC-MS/MS was performed using a Thermo Finnigan LC-MS/MS analyzer (ThermoFinnigan Quantum TSQ (S/N: TQU00655)) with an atmospheric pressurechemical ionization (APCI) source used as the detector. Liquidchromatography was performed with a Cohesive Technologies Aria TX-4(S/N: SJCTX409) LC system with Waters Symmetry C18 5 μm 4.6×50 mmcolumns. The multiplexed analytical HPLC system uses a C18 solid phasewith an isocratic separation using 100% methanol as the mobile phase.The autosampler injected 50 μL of extracted sample supernatant onto theHPLC column. After the analytes eluted and the detector window completedacquisition, the system was washed with 85% Mobile phase A and thenre-equilibrated with Mobile phase B for a run time of 5 minutes. Mobilephase A was 0.1% formic acid in HPLC-grade water and mobile phase B was100% methanol.

The flow of liquid solvent from the HTLC entered the heated nebulizerinterface of the Thermo Finnigan LC-MS/MS analyzer. The solvent/analytemixture was first converted to vapor in the heated tubing of theinterface. The analytes, contained in the nebulized solvent, wereionized (a positive charge added) by the corona discharge needle of theinterface, which applies a large voltage to the nebulizedsolvent/analyte mixture. The ions pass through the orifice of theinstrument and enter the first quadrupole. Quadrupoles 1 and 3 (Q1 andQ3) are mass filters, allowing selection of ions based on their mass tocharge ratio (m/z). Quadrupole 2 (Q2) is the collision cell, where ionsare fragmented.

The first quadrupole of the mass spectrometer (Q1) selected formolecules with the mass to charge ratios of (protonated and dehydrated)25OHD₂, 25OHD₃ and ⁶D-25OHD₃. Ions with these m/z ratios (see tablebelow) were allowed to pass into the collision chamber (Q2), whileunwanted ions with any other m/z collide with the sides of thequadrupole and are eliminated. Ions entering Q2 collide with neutralArgon gas molecules and fragment. This process is called CollisionActivated Dissociation (CAD). The fragment ions generated are passedinto quadrupole 3 (Q3), where the fragment ions of 25OHD₂, 25OHD₃ and⁶D-25OHD₃ were selected (see table below) and other ions are eliminated.The following mass transitions were used for detection and quantitationduring validation: TABLE 1 Mass transitions for selected vitamin Dmetabolites Compound Precursor Ion (m/z) Fragment Ions (m/z) 25OHD₂395.30 179.10, 251.30, 209.20 25OHD₃ 383.16 211.35 ⁶D-25OHD₃ 389.20211.30

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods.

Area ratios of the analyte and internal standard (Hexadeuterated25-Hydroxyvitamin D3, ⁶D-25OHD₃) peaks were used to constructcalibration curves, which were then used to calculate analyteconcentrations. Using the calibration curves, the concentrations of25OHD₂ and 25OHD3 were quantitated in the patient samples.

Example 2 Intra-Assay and Inter-Assay Precision

Three levels of spiked serum were made from patient pools (Medium andHigh Pools) and stripped serum from Golden West Biologicals, Product#SP1070 (Low Pool). Each pool was spikes with stock solutions of 25OHD₂and/or 25OHD₃ to bring analyte concentrations to the desired levels. Thetarget concentrations of the control pools used for these determinationswere: Low; 20-25 ng/mL, medium; 45-55 ng/mL and High; 100-110 ng/mL foreach analyte. Twenty aliquots from each level were analyzed in a singleassay using the LC-MS/MS protocols described in example 1. The followingprecision values were determined: TABLE 2 Intra-Assay Variation:25-Hydroxyvitamin D₂ (25OHD₂) Low Medium High 092804-L 092804-M 092804-H 1 26.5 46.4 103.3  2 23.2 51.1 96.7  3 23.1 52.4 107.8  4 21.6 50.3104.5  5 26.3 47.5 96.2  6 25.1 54.4 98.5  7 25.9 54.6 100.0  8 21.950.1 110.1  9 23.4 50.8 97.6 10 23.5 53.2 105.1 11 22.2 52.9 105.9 1224.0 54.6 94.5 13 26.2 49.4 93.4 14 24.1 59.0 113.0 15 25.8 52.9 112.416 23.9 59.2 113.4 17 29.5 52.4 107.7 18 24.2 50.0 115.5 19 19.8 53.5114.9 20 26.3 60.2 126.6 Average (ng/mL) 24.3 52.7 105.9 Std Dev 2.2 3.68.6 CV (%) 9.0 6.9 8.1

TABLE 3 Intra-Assay Variation: 25-Hydroxyvitamin D₃ (25OHD₃) Low MediumHigh 092804-L 092804-M 092804-H  1 22.7 43.6 99.1  2 22.4 45.3 93.5  322.4 50.7 98.2  4 21.0 40.1 95.9  5 21.8 41.5 82.0  6 20.8 42.2 97.4  722.9 50.1 96.0  8 19.0 42.0 106.7  9 21.8 44.2 96.6 10 23.4 49.5 94.9 1121.8 46.5 97.9 12 20.7 49.9 87.1 13 25.4 44.7 85.5 14 24.5 48.0 101.5 1525.1 45.8 101.5 16 22.5 52.0 104.7 17 29.2 45.9 107.7 18 19.5 49.3 107.619 18.1 49.6 109.4 20 24.8 49.3 116.1 Average (ng/mL) 22.5 46.5 99.0 StdDev 2.5 3.5 8.4 CV (%) 11.2 7.5 8.5

The spiked serum pools described above were also analyzed to determineinter-assay precision. Four aliquots from each level were analyzed overfive different assays using the LC-MS/MS protocols described inexample 1. The following precision values were determined: TABLE 4Inter-Assay Variation: 25-Hydroxyvitamin D₂ (25OHD₂) Low Medium High092804-L 092804-M 092804-H  1 26.5 46.4 103.3  2 23.2 51.1 96.7  3 23.152.4 107.8  4 21.6 50.3 104.5  5 26.3 47.5 96.2  6 25.1 54.4 98.5  725.9 54.6 100.0  8 21.9 50.1 110.1  9 23.4 50.8 97.6 10 23.5 53.2 105.111 22.2 52.9 105.9 12 24.0 54.6 94.5 13 26.2 49.4 93.4 14 24.1 59.0113.0 15 25.8 52.9 112.4 16 23.9 59.2 113.4 17 29.5 52.4 107.7 18 24.250.0 115.5 19 19.8 53.5 114.9 20 26.3 60.2 126.6 Average (ng/mL) 24.352.7 105.9 Std Dev 2.2 3.6 8.6 CV (%) 9.0 6.9 8.1

TABLE 5 Inter-Assay Variation: 25-Hydroxyvitamin D₃ (25OHD₃) Low MediumHigh 092804-L 092804-M 092804-H  1 22.7 43.6 99.1  2 22.4 45.3 93.5  322.4 50.7 98.2  4 21.0 40.1 95.9  5 21.8 41.5 82.0  6 20.8 42.2 97.4  722.9 50.1 96.0  8 19.0 42.0 106.7  9 21.8 44.2 96.6 10 23.4 49.5 94.9 1121.8 46.5 97.9 12 20.7 49.9 87.1 13 25.4 44.7 85.5 14 24.5 48.0 101.5 1525.1 45.8 101.5 16 22.5 52.0 104.7 17 29.2 45.9 107.7 18 19.5 49.3 107.619 18.1 49.6 109.4 20 24.8 49.3 116.1 Average (ng/mL) 22.5 46.5 99.0 StdDev 2.5 3.5 8.4 CV (%) 11.2 7.5 8.5

Example 3 Analytical Sensitivity: Limit of Detection and Limit ofQuantitation Studies

To determine the limit of detection of the assay, blank diluent wasanalyzed 17 times within a single run using the LC-MS/MS protocolsdescribed in example 1. The mean and standard deviation was thencalculated. The limit of detection was calculated as 2 SD above the meanof the blank peak area ratio based on a back calculation of peak arearatio against the calibration curve. The limits of detection were asfollows:

-   -   25OHD₂: 3.0 ng/mL    -   25OHD₃: 3.5 ng/mL

To determine the limit of quantitation atandard curves of 25OHD₂ and25OHD₃ were run in quadruplicate over five assays using the LC-MS/MSprotocols described in example 1. The ranges were 0, 2, 4, 8, 16, 32, 64and 128 ng/mL. The analyzed concentrations were pooled and thestatistical analysis was performed on values from 5 separate runs. Theresults of the study were as follows: TABLE 6 Limit of QuantitationStudy Results: 25-Hydroxyvitamin D₂ (25OHD₂) #1 #2 #3 #4 #5 Summary 0ng/mL −1.2 −1.3 −0.5 −1.2 −1.1 Average (ng/mL) −1.0 −1.1 −1.4 −0.9 −1.3−0.7 Standard Deviation 0.6 −1.1 −1.5 −0.8 NA −2.6 C of V (%) 59.0 0.4−1.7 −0.6 −0.5 −0.6 Accuracy (%) N/A 2 ng/mL 3.1 2.3 2.0 2.1 3.3 Average(ng/mL) 1.9 2.1 2.7 2.1 2.4 1.9 Standard Deviation 0.6 1.1 1.9 1.9 0.91.3 C of V (%) 33.7 1.2 1.1 2.1 1.8 1.4 Accuracy (%) 103.9 4 ng/mL 3.53.9 5.0 4.5 4.8 Average (ng/mL) 3.9 4.0 3.0 3.8 3.8 4.7 StandardDeviation 0.7 3.6 2.9 2.8 3.1 2.0* C of V (%) 17.1 4.1 4.6 4.5 4.4 3.7Accuracy (%) 101.7 8 ng/mL 10.2 9.1 9.1 8.8 9.8 Average (ng/mL) 8.6 7.88.1 7.9 8.4 9.0 Standard Deviation 0.8 8.6 8.3 7.4 8.4 7.5 C of V (%)9.3 10.2 8.4 8.0 8.1 8.6 Accuracy (%) 93.2 16 ng/mL  16.0 14.8 14.4 16.718.3 Average (ng/mL) 16.0 15.5 15.6 15.3 16.7 16.8 Standard Deviation1.1 16.6 16.7 16.8 15.8 15.2 C of V (%) 7.1 14.1 17.6 16.1 16.7 14.1Accuracy (%) 100.1 32 ng/mL  31.3 39.9* 29.9 33.2 32.7 Average (ng/mL)31.8 31.7 30.5 32.4 34.0 32.5 Standard Deviation 1.9 29.5 31.2 30.2 35.728.7 C of V (%) 6.0 32.9 34.7 32.4 30.8 29.2 Accuracy (%) 100.7 64ng/mL  66.5 62.2 68.5 62.6 68.8 Average (ng/mL) 64.6 66.6 67.8 67.3 58.961.5 Standard Deviation 3.2 64.4 61.4 63.7 63.5 61.3 C of V (%) 4.9 63.760.7 65.4 70.8 65.9 Accuracy (%) 99.1 128 ng/mL  125.1 128.2 123.4 127.8124.1 Average (ng/mL) 126.9 127.6 134.4 127.3 128.4 132.1 StandardDeviation 3.5 128.9 124.5 128.5 126.5 131.7 C of V (%) 2.8 126.1 119.7127.9 121.2 125.0 Accuracy (%) 100.8

TABLE 7 Limit of Quantitation Study Results: 25-Hydroxyvitamin D₃(25OHD₃) Day #1 Day #2 Day #3 Day #4 Day #5 (11/19/04-1) (11/19/04-2)(11/22/04-1) (11/23/04-1) (11/23/04-2) Summary 0 ng/mL −0.5 −0.9 −0.30.2 −0.6 Average (ng/mL) −0.7 −0.7 −1.3 0.3 −1.1 −1.0 Standard Deviation0.6 0.0 −1.0 −1.1 −1.3 0.6 C of V (%) 86.4 −0.3 −1.5 −1.1 −0.8 −1.0Accuracy (%) N/A 2 ng/mL 2.6 1.5 2.4 2.5 1.7 Average (ng/mL) 1.9 1.7 0.93.2 2.2 1.9 Standard Deviation 0.6 1.8 1.8 2.0 1.1 2.2 C of V (%) 31.41.3 2.4 1.5 1.1 2.5 Accuracy (%) 104.7 4 ng/mL 3.9 3.5 4.8 4.0 3.4Average (ng/mL) 3.8 3.0 4.1 3.1 3.9 3.8 Standard Deviation 0.8 4.5 3.72.9 3.5 2.4 C of V (%) 19.7 4.0 5.2 3.8 3.8 5.5 Accuracy (%) 104.1 8ng/mL 10.3 9.3 7.2 8.4 8.6 Average (ng/mL) 8.7 10.6 8.5 7.2 10.0 9.6Standard Deviation 1.1 7.4 10.3 9.0 9.4 8.4 C of V (%) 13.1 9.3 7.7 8.57.6 6.8 Accuracy (%) 91.9 16 ng/mL  15.9 15.6 16.2 18.6 17.1 Average(ng/mL) 16.0 13.8 16.3 14.0 17.2 15.3 Standard Deviation 1.4 15.6 16.115.1 18.8 16.1 C of V (%) 8.5 14.8 17.2 17.0 14.3 15.3 Accuracy (%) 99.932 ng/mL  31.1 35.8 29.6 32.8 28.0 Average (ng/mL) 31.7 30.8 29.9 31.833.0 32.8 Standard Deviation 1.9 31.6 30.9 29.5 35.7 31.2 C of V (%) 6.231.0 34.2 31.8 30.6 31.7 Accuracy (%) 101.0 64 ng/mL  65.9 64.6 64.464.8 67.8 Average (ng/mL) 64.8 67.4 62.9 62.4 60.7 57.2 StandardDeviation 3.1 68.9 64.2 62.1 64.0 64.7 C of V (%) 4.8 63.2 64.2 66.867.7 71.1 Accuracy (%) 98.8 128 ng/mL  128.9 124.5 126.4 125.3 122.8Average (ng/mL) 127.1 129.7 135.7 128.3 125.9 128.7 Standard Deviation4.7 125.5 123.3 127.2 127.7 135.4 C of V (%) 3.7 121.3 121.8 137.8 121.4124.1 Accuracy (%) 100.7

Example 5 Assay Reportable Range and Linearity

To establish the linearity of the vitamin D metabolite LC-MS/MS assay,the MultiProbe automated liquid handler robot independently constructedtwo standard curves by serially diluting a stock solution containing 128ng/mL 25OHD₂ and 128 ng/mL 25OHD₃ in 25OHD diluent (5% Bovine SerumAlbumin Fraction V dissolved in 0.01M PBS). The standard curve sampleswere analyzed using the LC-MS/MS protocols described in example 1. Thisprocess routinely produced standard curves with R² values of 0.99 orhigher for each analyte for the range of 4-128 ng/mL.

To determine whether patient samples can also be diluted in a linearfashion, a total of eight samples were serially diluted with 25OHDdiluent. Two samples were patient pools (Medium and High Control Pools),three were patient samples with high 25OHD₂ values and three werepatients with high 25OHD₃ values. All samples were analyzed using theLC-MS/MS protocols described in example 1. As shown in FIG. 1 and FIG.2, each sample diluted in a linear fashion (R²>0.98), demonstrating thesuitability of 25OHD diluent for diluting patient samples.

To demonstrate that elevated samples can be diluted into the linearrange of the assay, aliquots of 25OHD diluent were spiked to 512 ng/mLeach 25OHD₂ and 25HOD₃, then serially diluted to 8 ng/mL using the 25OHDdiluent. Each sample was extracted and run in duplicate using theLC-MS/MS protocols described in example 1. As shown in FIG. 3, each ofthese curves was linear (R²>0.99).

Example 6 Accuracy of LC-MS/MS Vitamin D Assay

The standards for 25OHD₂ and 25OHD₃ were quantified based upon theabsorbance of the concentrated (10-50 μg/mL) stock solutions in theultraviolet spectrum. The cis-triene chromophore present in all vitaminD compounds has a peak absorbance of 264 nm, which is dependent upon theanalyte concentration. The molar extinction coefficient of 18.3 mM⁻¹cm⁻¹was determined using purified, dessicated ergocalciferol andcholecalciferol and was used to determine the concentration of stocksolutions for 25OHD₂ and 25OHD₃.

To determine the ability to recover vitamin D metabolites from spikedserum samples, three patient pools of known concentrations were spikedwith two levels of 25OHD₂, 25OHD₃ and both 25OHD₂ and 25OHD₃ together.Each sample was extracted and run in duplicate using the LC-MS/MSprotocols described in example 1. The recovery was calculated bydividing the expected result by the observed result. TABLE 8 Recovery of25-hydroxylated vitamin D metabolites from spiked samples. 25OHD₂ 25OHD₃25OHD₂ 25OHD₃ (ng/mL) (ng/mL) (% Recovery) (% Recovery) Pool#1 58 51 — —Pool#1 + 20 ng/mL 25OHD₂ 75 52 104 — Pool#1 + 20 ng/mL 25OHD₃ 52 68 —105  Pool#1 + 20 ng/mL of both 72 68 109 105  Pool#1 + 50 ng/mL 25OHD₂104 50 105 — Pool#1 + 50 ng/mL 25OHD₃ 56 109 — 93 Pool#1 + 50 ng/mL ofboth 104 110 104 92 Pool#2 53 47 — — Pool#2 + 20 ng/mL 25OHD₂ 76 51  97— Pool#2 + 20 ng/mL 25OHD₃ 52 66 — 101  Pool#2 + 20 ng/mL of both 70 65105 103  Pool#2 + 50 ng/mL 25OHD₂ 107 53  96 — Pool#2 + 50 ng/mL 25OHD₃57 109 — 88 Pool#2 + 50 ng/mL of both 100 105 103 92 Pool#3 53 47 — —Pool#3 + 20 ng/mL 25OHD₂ 69 44 106 — Pool#3 + 20 ng/mL 25OHD₃ 55 75 — 90Pool#3 + 20 ng/mL of both 74 69  98 97 Pool#3 + 50 ng/mL 25OHD₂ 96 48107 — Pool#3 + 50 ng/mL 25OHD₃ 53 114 — 85 Pool#3 + 50 ng/mL of both 105113  98 85

Example 7 Comparison LC-MS/MS Vitamin D Metabolite Assay and RIAProcedures

A total of 1,057 patient samples were assayed using the LC-MS/MS methodsdescribed in example 1 and using an RIA commercially available fromDiaSorin and the data were compared. As shown in FIG. 4, for total25OHD, the R² value was 0.5082 with a slope of 0.9684.

Example 8 Cross-Reactivity Studies

Samples were prepared by diluting various commercially available vitaminD metabolites and analogues at a concentration of 100 ng/mL in 25OHDdiluent. Samples were extracted and run in duplicate using the LC-MS/MSmethods described in example 1. None of the tested compounds hadmeasurable signals detected in the 25OHD₂ and 25OHD₃ detection channels.TABLE 9 Cross-Reactivity of the LC-MS/MS method with various vitamin Danalogues and metabolites. Cross- Cross- Compound Reactivity ReactivityMass (Da) (25OHD₂) (25OHD₃) 25-Hydroxyvitamin D₂ 412 (100%) ND (25OHD₂)25-Hydroxyvitamin D₃ 400 ND (100%) (25OHD₃) Internal Standard(⁶D-25OHD₃) 406 ND ND Vitamin D₂ (Ergocalciferol) 396 ND ND Vitamin D₃(Cholecalciferol) 384 ND ND 1□,25(OH)₂D₂ 428 ND ND 1□,25(OH)₂D₃ 416 NDND 25,26(OH)₂D₃ 416 ND ND 1□(OH)D₂ (Doxercalciferol) 412 ND ND 1□(OH)D₃(Alfacalcidiol) 400 ND ND

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for determining the presence or amount of 25-hydroxyvitaminD in a biological sample, comprising: (a) ionizing said25-hydroxyvitamin D, if present in said sample; and (b) detecting thepresence or amount of said ion by mass spectrometry, wherein thepresence or amount of said ion is related to the presence or amount of25-hydroxyvitamin D in said test sample.
 2. The method of claim 1,wherein said sample is purified prior to said ionization step.
 3. Themethod of claim 2, wherein said purification is performed bychromatography.
 4. The method of claim 3, wherein said chromatography isliquid chromatography.
 5. The method of claim 4, wherein saidchromatography is high performance liquid chromatography (HPLC).
 6. Themethod of claim 1, wherein said sample or said 25-hydroxyvitamin D isnot subjected to gas-chromatography prior to said ionization step. 7.The method of claim 1, wherein said 25-hydroxyvitamin D is25-hydroxyvitamin D₃.
 8. The method of claim 7, wherein said ion has amass/charge ratio (m/z) of about 383.2.
 9. The method of claim 1,wherein said 25-hydroxyvitamin D is 25-hydroxyvitamin D₂.
 10. The methodof claim 9, wherein said ion has a mass/charge ratio (m/z) of about395.3.
 11. The method of claim 1, wherein said ionization step (a)comprises: (i) ionizing 25-hydroxyvitamin D, if present in said sampleto produce a 25-hydroxyvitamin D ion; (ii) isolating said25-hydroxyvitamin D ion by mass spectometry to provide a precursor ion;(iii) effecting a collision between the isolated precursor ion and aninert collision gas to produce at least one fragment ion detectable in amass spectrometer.
 12. The method of claim 11, wherein said25-hydroxyvitamin D is 25-hydroxyvitamin D₃.
 13. The method of claim 12,wherein said 25-hydroxyvitamin D₃ precursor ion has a mass/charge ratio(m/z) of about 383.16, and said 25-hydroxyvitamin D₃ fragment ion has anm/z of about 211.35.
 14. The method of claim 11, wherein said25-hydroxyvitamin D is 25-hydroxyvitamin D₂.
 15. The method of claim 14,wherein said 25-hydroxyvitamin D₂ precursor ion has a mass/charge ratio(m/z) of about 383.16, and said at least one 25-hydroxyvitamin D₂fragment ions comprise fragment ions with a m/z of 179.1, 209.20, and251.30.
 16. A method for determining the presence or amount of1,25-dihydroxyvitamin D₂ in a biological sample, comprising: (a)ionizing said 1,25-dihydroxyvitamin D₂, if present in said sample, toproduce an ion detectable by mass spectrometry; and (b) detecting thepresence or amount of said ion by mass spectrometry, wherein thepresence or amount of said ion is related to the presence or amount of1,25-dihydroxyvitamin D₂ in said test sample;
 17. The method of claim16, wherein said sample is purified prior to said ionization step. 18.The method of claim 17, wherein said purification is performed bychromatography.
 19. The method of claim 18, wherein said chromatographyis liquid chromatography.
 20. The method of claim 19, wherein saidchromatography is high performance liquid chromatography (HPLC).
 21. Themethod of claim 16, wherein said sample or said 25-hydroxyvitamin D isnot subjected to gas-chromatography prior to said ionization step. 22.The method of claim 16, wherein said ionization step (a) comprises (i)ionizing 1,25-dihydroxyvitamin D₂, if present in said sample to producea 1,25-dihydroxyvitamin D₂ ion; (ii) isolating said1,25-dihydroxyvitamin D₂ ion by mass spectometry to provide a precursorion; (iii) effecting a collision between the isolated precursor ion andan inert collision gas to produce at least one fragment ion detectablein a mass spectrometer.
 23. A method for determining the presence oramount of two or more vitamin D metabolites in a test sample in a singleassay, said method comprising: (a) ionizing said two or more vitamin Dmetabolites, if present in said sample, to generate ions specific foreach of said two or more vitamin D metabolites; and (b) detecting thepresence or amount of said ions by mass spectrometry, wherein thepresence or amount of said ions is related to the presence or amount ofsaid vitamin D metabolites in said test sample.
 24. The method of claim23, wherein said sample is purified prior to said ionization step. 25.The method of claim 24, wherein said purification is performed bychromatography.
 26. The method of claim 25, wherein said chromatographyis liquid chromatography.
 27. The method of claim 26, wherein saidchromatography is high performance liquid chromatography (HPLC).
 28. Themethod of claim 23, wherein said sample or said two or more vitamin Dmetabolites are not subjected to gas-chromatography prior to saidionization step.
 29. The method of claim 23, wherein said vitamin Dmetabolites comprise 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂. 30.The method of claim 29, wherein said 25-hydroxyvitamin D₃ ion has amass/charge ratio (m/z) of about 383.16 and said 25-hydroxyvitamin D₂ion has a mass/charge ratio (m/z) of about 395.30.
 31. The method ofclaim 23, wherein said mass spectrometry analysis comprises tandem massspectrometry;
 32. The method of claim 31, wherein said vitamin Dmetabolites comprise 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂. 33.The method of claim 32, wherein the mass/charge ratio (m/z) for the25-hydroxyvitamin D₃ precursor ion is about 383.16, the m/z for the25-hydroxyvitamin D₃ fragment ion is about 211.35, the m/z for the25-hydroxyvitamin D₂ precursor ion is about 395.30 and the m/z for the25-hydroxyvitamin D₂ fragment ions are about 179.1, 209.20, 251.30. 34.The method of claim 23 or claim 31, wherein said two or more vitamin Dmetabolites are vitamin D metabolites selected from the group consistingof 25-hydroxyvitamin D₃, 25-hydroxyvitamin D₂, 1,25-dihydroxyvitamin D₃and 1,25-dihydroxyvitamin D₂
 35. The method of claim 34, wherein said ofvitamin D metabolites comprise 1,25-dihydroxyvitamin D₃ and1,25-dihydroxyvitamin D₂.